This article demonstrates that GVHD is characterized by neovascularization, which is mainly driven by vasculogenesis, as opposed to angiogenesis. We identify inhibition of neovascularization as a novel therapeutic concept to simultaneously reduce inflammation and the growth of tumors: the VE-cadherin monomer–specific antibody E4G10 simultaneously inhibited GVHD and tumor growth, resulting in reduction of both GVHD-related mortality and tumor-related mortality, leading to improved long-term survival of tumor-bearing allo-BMT recipients. We conclude that targeting of neovasculature provides a unique means to address the two major complications of allogeneic hematopoietic stem cell transplantation (GVHD and tumor relapse) with a single therapeutic strategy.
A key feature of our study is that it used the MHC disparity of donor and host to separately quantify vasculogenesis and angiogenesis by flow cytometry (). Our MHC class I–mismatched model (B6→BALB/c), in which C57BL/6 BM cells and C57BL/6 T cells were transferred into lethally irradiated BALB/c recipients, enabled us to easily distinguish vasculogenesis from angiogenesis by H-2b (C57BL/6) and H-2d
(BALB/c) expression. We found that only the number of donor BM-derived ECs increased during GVHD, whereas the number of host resident tissue ECs remained constant. The predominant role that donor ECs play in the formation of neovasculature after allo-BMT is not surprising because of the negative effect that lethal doses of irradiation have on host EC function. The inhibition of angiogenesis by irradiation is well known and has been demonstrated by many groups (30
). Even relatively low doses of irradiation, such as 2 and 4 Gy, potently inhibit EC proliferation (38
). It has been shown that irradiation doses similar to those used clinically are sufficient to inhibit EC function in allo-BMT recipients (41
). Transplanted tumors grow more slowly when the tissue around them becomes irradiated (42
). This effect is termed the “tumor bed effect” and is explained by the vascular damage caused by the radiation leading to inhibition of host angiogenesis (42
). The tumor bed effect is used experimentally to block angiogenesis, favoring vasculogenesis, in experimental tumor models (44
The role of vasculogenesis and EPCs in neovascularization has been a topic of considerable discussion over the past decade (1
). Some controversy regarding the contribution of EPCs to neovasculature was based on the use of nonselected BM cells in previous studies, which could have resulted in the contribution of non-EPC hematopoietic cells of donor origin. We therefore performed experiments with purified GFP-expressing EPCs of donor origin and found recruitment to neovasculature (, and ; Supplementary Figure 1
and Supplementary Movies 1
, available online). Furthermore, we quantified vasculogenesis with another method: using donor–host markers in flow cytometry, we found that donor BM–derived ECs contribute to host neovasculature during GVHD (). Thus, we used two independent methods and consistently found contribution of donor BM-derived EPCs to host neovasculature during GVHD.
We have demonstrated that the inhibition of neovascularization with an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin can be used therapeutically to inhibit inflammation during GVHD. The main mechanism is likely to be the inhibition of vasculogenesis in GVHD target organs, which leads to a reduced recruitment of proinflammatory cells migrating via the blood vessels to inflammatory sites. We cannot exclude that donor BM-derived EPCs exhibit some paracrine proinflammatory functions, which could explain inhibition of inflammation after depletion of EPCs. However, since serum cytokine and chemokine levels as well as the number of circulating inflammatory cells were not reduced as a result of E4G10 treatment, this possibility appears less likely. There are potential clinical implications of our findings for a broad variety of inflammatory diseases. Previous findings have shown that EPCs are recruited from peripheral blood to neovasculature during inflammation in several inflammatory diseases, including asthma (50
) and rheumatoid arthritis (51
), so justify studies on the efficacy of this therapeutic approach in inflammatory diseases, other than GVHD.
E4G10 specifically targets VE-cadherin monomers on EPCs as opposed to VE-cadherin trans-dimers in established vasculature. We know of no direct evidence that cell surface VE-cadherin monomers are more abundant on inflamed vessels compared with normal vessels. This is in keeping with our findings that E4G10 did not bind specifically to vasculature in GVHD target organs. However, we cannot completely rule out that E4G10 binds to host endothelium at sites of inflammation and, therefore, could have some effects on host angiogenesis. In tumor vasculature, Liao et al. (52
) found that E4G10 stains a subset of tumor vessels. However, because angiogenesis was not increased in our GVHD models with lethally irradiated ECs (, C) and administration of E4G10 had no effect on angiogenesis (), a major effect of E4G10 on host vasculature appeared to be very unlikely in our study. In addition, the findings of Liao et al. are in contrast to later studies by Nolan et al. (12
), who published compelling data showing that E4G10 recognized only monomeric VE-cadherin expressed exclusively on EPCs and not the dimerized form in host vessels. Importantly, we observed 1) that GVHD is characterized by neovascularization and 2) that the administration of E4G10 leads to simultaneous benefits with respect to GVHD and tumor growth. This observation—and its clinical relevance—is independent of the question of whether E4G10 binds specifically to EPCs or whether it also binds to host vasculature.
In our study, the inhibition of neovascularization with E4G10 reduced the intensity of GVHD but did not completely prevent it. It would be of great interest to determine whether the dosing schedule of anti–VE-cadherin antibodies could be further optimized to enhance GVHD prevention. In the clinical setting, the inhibition of neovascularization most likely would be combined with current standard therapies to prevent or treat GVHD, such as T-cell depletion and immunosuppression.
We found that VEGF gene expression was not increased during GVHD (Supplementary Table 6
, available online), which suggests that VEGF might not be the dominant proangiogenic factor during GVHD in our models. Interestingly, Min et al. (53
) found in a clinical study that low VEGF serum levels after allo-SCT are associated with high mortality and with an exacerbated severity of acute GVHD. These results are in agreement with a recent publication that links VEGF gene polymorphisms that lead to lower production of VEGFA with increased incidence of acute GVHD (54
). To test whether VEGF could be used as a therapeutic target during GVHD, we used anti-VEGFR1/anti-VEGFR2 antibodies after allo-BMT and found an inhibitory effect of on hematopoietic reconstitution leading to early death of allo-BMT recipients. In another GVHD model, a higher and more rapid mortality was seen in allo-BMT recipients treated with anti-VEGF peptide (53
). These results suggest that the use of anti-VEGF strategies for prevention of GVHD after allo-BMT may not be effective and may potentially inhibit hematopoietic reconstitution. Taken together, it is currently not possible to draw final conclusions regarding the role of VEGF in the regulation of vasculogenesis and angiogenesis during GVHD.
Recently, we and other investigators demonstrated that vasculogenesis contributes to tumor vasculature and that depletion of EPCs leads to inhibition of primary as well as metastatic growth of solid tumors in certain models (8
). This study extends these findings to tumors after allo-BMT and to hematological malignancies: We found that E4G10 administration resulted in inhibition of lymphoma and AML growth in vivo. Our results are in agreement with recent clinical data suggesting a critical role for neovascularization not only in solid tumors but also in hematological malignancies (55
). However, the inhibition of tumor growth that we observed in hematological malignancies as a result of E4G10 administration was rather modest and model-dependent. A possible approach to enhance the efficacy of E4G10 toward hematological malignancies in future studies would be the optimization of the dosing schedule (eg, a longer treatment duration) and the use of E4G10 bound to alpha particles (eg, Ac-E4G10), which deliver potent short-ranged radiation and have been successfully used in solid tumor models (14
). Interestingly, we found that the benefit of E4G10 on tumor growth seemed to be stronger in the presence of donor T cells than in their absence. One possible explanation is that administration of E4G10 may lead to a normalization of tumor vasculature, increasing the blood flow, and leading to a more effective recruitment of tumor-reactive T cells to the tumor tissue (1
Our study has some limitations. First, we showed that administration of the E4G10 antibody leads to depletion of EPCs and to inhibition of neovascularization during inflammation as well as tumor growth. However, with the methods applied, it is not possible to prove that the mechanism of the E4G10 action is exclusively mediated by its effects on EPCs (as opposed to an unknown effect of E4G10 on other cells or structures in the body). Second, several endpoints in our experiments, such as the histological scoring and clinical scoring of GVHD, are subjective. Therefore, we blinded the evaluators who performed the histological scoring. For practical reasons, it was not possible to perform blinded clinical scoring. However, to minimize bias, those persons with direct interests in the project were excluded from clinically scoring and killing the mice
In conclusion, this study demonstrates that GVHD is characterized by increased neovascularization and identifies the inhibition of neovascularization with an antibody against VE-cadherin monomers as a novel therapeutic concept to simultaneously ameliorate GVHD and tumor growth.